Abstract

The present study sought to determine whether chemical destruction of peripheral catecholaminergic fibers with 6-hydroxydopamine (6OHDA) attenuates vasopressin (VP) and oxytocin (OT) secretion stimulated by hemorrhage, hypotension, and hyperosmolality. Rats received 6OHDA (100 mg/kg iv) or vehicle (1 ml/kg iv) on days 1 and 7, and experiments were performed on day 8. Serial hemorrhage (4 samples of 2 ml per 300 g body wt at 10-min intervals) increased plasma VP and OT levels in both groups; however, the increase in plasma VP and OT levels was significantly attenuated in 6OHDA-treated vs. control rats despite a significantly lower mean arterial blood pressure. Similarly, the increase in plasma VP and OT levels in response to hypotension produced by the selective arteriolar vasodilator diazoxide was significantly attenuated in 6OHDA-treated rats. In marked contrast to hemorrhage and hypotension, hyperosmolality produced by an infusion of 1 M NaCl (2 ml/h iv) stimulated increases in plasma VP and OT levels that were not different between 6OHDA-treated and control rats. In a parallel set of experiments, intravenous 6OHDA treatment reduced dopamine-β-hydroxylase immunoreactivity in the posterior pituitary but had no substantial effect in the hypothalamic paraventricular and supraoptic nuclei. In each experiment, the pressor response to tyramine (250 μg/kg iv) was significantly attenuated in 6OHDA-treated rats, thereby confirming that 6OHDA treatment destroyed sympathetic catecholaminergic fibers. Collectively, these findings suggest that catecholaminergic fibers located outside the blood-brain barrier contribute to VP and OT secretion during hemorrhage and arterial hypotension.

blood pressure

catecholamines

osmolality

blood volume

baroreceptor

magnocellular neurosecretory neurons of the hypothalamic supraoptic and paraventricular nuclei synthesize vasopressin (VP) and oxytocin (OT) and release these hormones directly into the circulation from axon terminals located in the posterior pituitary. VP promotes water reabsorption in the kidney by altering membrane permeability of the distal tubules and collecting ducts and, at higher levels, produces vasoconstriction (6). OT, in addition to its well-known roles in lactation and parturition, stimulates natriuresis in rats (33) and modulates hypotension-evoked renin secretion (13). Together, these hormones act in concert with other physiological responses (e.g., ingestion of water and salt, changes in autonomic nervous system activity) to regulate body fluid homeostasis under a variety of circumstances, including alterations in blood volume, arterial blood pressure, or plasma osmolality (Posmol).

The secretion of VP and OT is influenced by a number of neuroactive substances acting within the supraoptic and paraventricular nuclei (25). For example, noradrenergic fibers originating from the central nervous system innervate both VP and OT magnocellular neurons (32). Functional studies suggest that these noradrenergic inputs relay visceral information from the hindbrain to affect the excitability of putative VP and OT neurons and subsequent release of VP and OT (4, 11, 25, 26). On the other hand, VP and OT secretion may also be modulated by substances acting directly at the level of magnocellular nerve terminals in the posterior pituitary. For example, the posterior pituitary is innervated by catecholaminergic fibers that likely arise from both central and peripheral origins (3, 10). With regard to the latter, removal of the superior cervical ganglion decreases posterior pituitary norepinephrine (1, 19), and electrical stimulation of the superior cervical ganglion increases VP and OT secretion (16, 17). Recently, we reported that intravenous infusion of the α1-adrenergic agonist phenylephrine at doses that do not cross the blood-brain barrier potentiate plasma VP and OT levels in hyperosmotic rats (29). Collectively, these observations suggest that peripheral catecholaminergic inputs to the posterior pituitary enhance VP and OT secretion.

In the present study, we sought to determine whether the catecholaminergic fibers located outside the blood-brain barrier contributed to VP and OT secretion during various physiological challenges. Peripheral catecholaminergic fibers were destroyed by intravenous administration of the neurotoxin 6-hydroxydopamine (6OHDA), and VP and OT secretion was stimulated by hemorrhage, hypotension, or hyperosmolality.

METHODS

Animals

Adult male Sprague-Dawley rats (Harlan Laboratories, Indianapolis, IN) weighing 300–400 g were housed individually in a temperature-controlled room (22–23°C) with a 12:12-h light-dark-cycle (lights on at 8:00 AM). Tap water and Purina Laboratory Chow (No. 5001) were available ad libitum except where noted. Body weights were not different between groups in any experiment. All experimental and surgical procedures conform with the APS Guiding Principles in the Care and Use of Animals and were approved by the Institutional Animal Care and Use Committee at the University of Kentucky and University of Pittsburgh.

Chemical Sympathectomy

Chemical sympathectomy was performed in rats with intravenous administration of 6OHDA (100 mg/kg) on days 1 and 7, as described previously (7, 18). On day 1, rats were anesthetized with halothane (2–3% in 100% O2), and 6OHDA followed by 0.3 ml isotonic saline was given through a Silastic catheter inserted into the right jugular vein. The catheter was removed, the jugular vein tied off, and the incision closed with suture. Rats received an injection of antibiotic (Dual-cillin, 30,000 U im) and were returned to home cages. On day 7, catheters were implanted in the left femoral artery (Silastic or Microrenthane tubing, 0.012 in. ID and 0.025 in. OD, Braintree Scientific, Braintree, MA) and vein (Silastic, 0.025 in. ID and 0.037 in. OD, Fisher Scientific, Pittsburgh, PA) while rats were anesthetized with halothane (2–3% in 100% O2). These catheters were tunneled subcutaneously to exit between the scapulae and were filled with heparinized saline (arterial, 1,000 U/ml; venous, 40 U/ml). Rats were fitted with an infusion harness (Harvard Apparatus) that allowed the catheters to pass outside the cage while protected by a steel spring. Then, rats were injected with 6OHDA through the femoral venous catheter. On both days, control rats received 0.1 M ascorbic acid vehicle (1 ml/kg iv). Rats received an injection of antibiotic (Dual-cillin, 30,000 U im), and experiments began the next day.

Effect of 6OHDA Treatment on VP and OT Secretion

At least 1 h before experiments began, rats were weighed and returned to the home cage without food and water. Arterial blood pressure was recorded by connecting the arterial line to a Statham pressure transducer (Grass Instruments, Quincy, MA) and a polygraph chart recorder (model 7; Grass Instruments). The pulsatile arterial blood pressure signal was filtered electronically to obtain mean arterial blood pressure (MAP). Heart rate (HR) was obtained through a tachograph (Grass Instruments, Model 7P44) triggered by the pulsatile arterial blood pressure signal.

Tyramine test.

The completeness of the chemical sympathectomy was assessed by the magnitude of the pressor response to tyramine (250 μg/kg iv) in both 6OHDA-treated and control rats. Tyramine stimulates vasoconstriction and increases blood pressure by promoting endogenous norepinephrine release from sympathetic nerve terminals (7, 12). Each rat was tested three times separated by 5 min, and the peak changes in MAP were averaged. Hemorrhage, hypotension, or hyperosmolality protocols began 1 h after the last tyramine injection.

Hemorrhage.

Rats were hemorrhaged serially as described previously (23, 24). After a 20-min recording of baseline MAP and HR, four successive blood samples (2 ml/300 g body wt over 1 min) at 10-min intervals were collected from the arterial line into microcentrifuge tubes containing heparin (20 U/1.5 ml blood). Samples were centrifuged immediately (10,000 g, 1 min), and the plasma was stored at −80°C until VP and OT levels were determined by radioimmunoassay, as described below. Posmol was measured from two 20-μl aliquots by freezing point depression using a micro-osmometer (model 3360; Advanced Instruments; Norwood, MA).

Hypotension.

A second group of 6OHDA-treated and control rats received an injection of the arteriolar vasodilator diazoxide (DZX; 15 mg/kg iv) to decrease MAP. Because destruction of sympathetic nerves would be expected to exaggerate the hypotensive effect of DZX, an additional group of control rats received a larger dose of DZX (25 mg/kg iv) to produce similar degrees of hypotension as those observed in 6OHDA-treated rats receiving DZX (15 mg/kg iv). The dose of DZX will be referred to as DZX plus the respective dose (i.e., DZX15 or DZX25). Blood samples (2.0 ml) were collected from the arterial line as described above at 5 min before injection of DZX, and 10 and 30 min afterward. Posmol was determined as described above. In this and subsequent experiments, the first blood sample was replaced by an equal volume of isotonic saline, whereas subsequent blood samples were replaced with red blood cells from the previous sample resuspended in heparinized saline (40 units/ml).

Hyperosmolality.

Another group of 6OHDA-treated and control rats were infused with hypertonic saline (HS; 1 M NaCl) at a rate of 2 ml/h iv for 120 min to raise Posmol. Blood samples (2.0 ml) were collected from the arterial line into a microcentrifuge, as described above, at 5 min before initiation of the 1 M NaCl infusion, and 60 and 120 min after the start of the infusion. Posmol was determined as described in the experiments in Hemorrhage.

Determination of Plasma VP and OT Levels

Plasma VP and OT levels were determined by radioimmunoassay, as described previously (22, 29). Briefly, samples were extracted using C18 Sep-Pak Vac Cartridges (1 ml, 50 mg; Waters, Milford, MA), and the extract was frozen, dried using a Speed Vac (Savant Instruments), and reconstituted in buffer (50 mM NaPO4, 25 mM EDTA, 0.9% NaCl, 0.5% BSA, 0.1% sodium azide); 150-μl aliquots were used for radioimmunoassays. Samples were incubated for 16–24 h at 4°C with a rabbit polyclonal antibody to either VP (final dilution 1:300,000) or OT (1:450,000). The characteristics of these antibodies, which were generously donated by Dr. J. Fernstrom (Pittsburgh, PA), have been described previously (9). Then, samples were incubated for 16 h at 4°C with ∼3,200 counts/min of 125I-labeled VP or OT (New England Nuclear-DuPont, Boston, MA). Subsequently, antibodies were precipitated using a second antibody procedure (22), and tubes were centrifuged (3,000 g, 25 min), the supernatant was aspirated, and the remaining pellets were counted in a gamma counter (1470 Wizard; Wallac, Gaithersburg, MD). Values of VP and OT were calculated from standard curves generated with known values of synthetic VP or OT (Bachem, Torrance, CA) that were extracted identically to plasma samples. Duplicate plasma VP samples and single plasma OT samples were analyzed, and values are expressed as picograms per milliliter of plasma.

To determine whether 6OHDA treatment destroyed catecholaminergic innervation of the posterior pituitary without affecting the innervation of structures inside the blood-brain barrier, we examined dopamine-β-hydroxylase (DβH) immunoreactivity in the posterior pituitary and hypothalamic structures in 6OHDA and vehicle-treated rats. A separate group of rats were treated with 6OHDA (n = 3) or vehicle (n = 3) on day 1 and 7, and the pressor response to tyramine (250 μg/kg iv) was examined as described above. Then, rats were deeply anesthetized with Inactin (100 mg/kg iv) and perfused transcardially with 100 ml isotonic saline followed by 250 ml of 4% paraformaldehyde in 0.1 M PBS (4°C). Brains and pituitaries were removed, postfixed overnight in 4% paraformaldehyde at 4°C, and immersed in 30% sucrose for 2–3 days.

Pituitaries were sectioned at 20 μm using a cryostat, mounted on slides in a series of 1 in 8, and stored at −20°C. Tissue sections were then rinsed with 0.1 M PBS and 0.1% Triton X-100, incubated with 0.3% Triton X-100 in 100% methanol for 20 min to quench endogenous peroxidase activity, and blocked with 0.1 M PBS, 0.1% Triton X-100, and 10% normal donkey serum (blocking solution) for 1 h. Sections were incubated overnight at 4°C with a monoclonal mouse anti-DβH antibody (1:500, MAB308; Chemicon, Temecula, CA) in blocking solution followed by a 1-h incubation at room temperature with a Cy3 donkey anti-mouse IgG (1:100, Jackson ImmunoResearch Laboratories, West Grove, PA). Forebrains were sectioned at 30 μm using a cryostat and collected into 0.1 M PBS at 4°C in a one in three series. Then, free-floating sections were rinsed with 0.1 M PBS, and incubated with a monoclonal anti-DβH antibody (1:500 MAB308; Chemicon) at 4°C for 48 h followed by an overnight incubation in a Cy3 donkey anti-mouse IgG (1:100, Jackson Immunoresearch Laboratories). Sections were mounted on slides and coverslipped with Cytoseal 60 (Fisher Scientific).

Statistical Analysis

All data are expressed as means ± SE. MAP and HR were analyzed by a two-way ANOVA with repeated measures (Systat 10.2, Systat Software, Point Richmond, CA). When significant F values were obtained for the group factor, independent t-tests corrected with a layered Bonferroni analysis were performed. When significant F values were obtained for the time factor, an ANOVA with repeated measures was performed followed by paired t-tests corrected with a layered Bonferroni analysis. Plasma VP and OT concentrations were log transformed and analyzed as described for MAP and HR. Posmol were analyzed as described for MAP and HR. For hypotension experiments, a linear regression analysis was performed between plasma VP and OT levels and the level of MAP (Sigma Plot 2000, SPSS). A P value of <0.05 was significant in all statistical tests.

DβH immunoreactivity was ranked by two experimenters blind to the treatment group using the following scale: 0, no labeling; 1, sparse; 2, light moderate; 3, heavy moderate; and 4, dense. Scores were averaged and analyzed by a Mann-Whitney U-test. Scores for a section did not vary more than one unit on the scale. Tissue sections were sampled from several brain regions, including the subfornical organ, dorsal and ventral median preoptic nucleus, organum vasculosum of the lamina terminalis, the supraoptic nucleus, hypothalamic paraventricular nucleus, and the posterior pituitary. One section per animal was scored for each area except for the posterior pituitary that was scored from two sections per animal. In addition, the hypothalamic paraventricular nucleus was sampled from three rostrocaudal levels, as described previously (27, 28, 30): level 1 was the most rostral and consisted of a ventrally located magnocellular division; level 2 displayed a prominent and laterally positioned posterior magnocellular division and both dorsal and ventrolateral parvocellular divisions; level 3 was the most caudal and consisted of the lateral and medial parvocellular divisions. Digital images were taken of each area using a Nikon Eclipse TE2000-E microscope connected to a Spot camera (Spot RT Slider, Diagnostic Instruments, Sterling Heights, MI) using Spot Imaging Software (version 3.5).

Effect of 6OHDA Treatment on Hemorrhage-Evoked Increase in Plasma VP and OT Levels

Serial hemorrhage significantly increased plasma VP and OT levels from baseline levels at 20 and 30 min in both 6OHDA-treated and control rats (Figs. 1, A and B). However, the hemorrhage-evoked increase in plasma VP and OT levels was significantly attenuated in 6OHDA-treated rats compared with control rats at both times (Fig. 1, A and B). Baseline plasma VP and OT levels did not differ between groups. The attenuated increase in plasma VP and OT levels of 6OHDA-treated rats occurred despite a significantly lower MAP compared with control rats between 2 and 15 min (Fig. 1C). In fact, MAP of 6OHDA-treated rats fell significantly below baseline values immediately after the first blood withdrawal, whereas MAP of control rats did not significantly drop until the second blood withdrawal. In both 6OHDA-treated and control rats, hemorrhage produced a biphasic response in HR with an initial tachycardia followed by a significant bradycardia. Although baseline HR were not different between groups (6OHDA: 383 ± 5 bpm; control: 386 ± 11 bpm), the magnitude of the tachycardic response was significantly attenuated in 6OHDA-treated vs. control rats (6 min values: 34 ± 8 vs. 70 ± 14 bpm, respectively; P < 0.05); the magnitude of the bradycardia was not different between groups (−91 ± 19 bpm vs. −115 ± 33 bpm). Posmol was not significantly different between the two groups at any time (Table 1).

Mean ± SE of plasma vasopressin (VP; A) and oxytocin (OT; B) levels and mean arterial blood pressure (MAP; C) of 6-hydroxydopamine (6OHDA)-treated and control rats that were hemorrhaged serially. An arrow indicates the time when a blood sample was withdrawn. Hemorrhage significantly increased plasma VP and OT levels above baseline levels in both groups at 20 and 30 min (P < 0.05). However, both plasma VP and OT levels of 6OHDA-treated rats were significantly lower than those of control rats at 20 and 30 min. C: MAP of 6OHDA-treated rats was significantly lower than those values of control rats between 2 and 15 min. *Significant difference between 6OHDA-treated and control rats (P < 0.05).

Plasma osmolality of 6OHDA-treated and control rats that were hemorrhaged serially or received an injection of DZX

Effect of 6OHDA Treatment on Hypotension-Evoked Increase in Plasma VP and OT Levels

Administration of DZX15 significantly decreased MAP in both 6OHDA-treated and control rats (Fig. 2C). However, 6OHDA-treated rats displayed a significantly lower MAP than control rats receiving DZX15, which would provide a greater stimulus for VP and OT secretion. Therefore, to produce similar levels of MAP between 6OHDA-treated and control rats, a larger dose of DZX was administered to a separate group of control rats. MAP of 6OHDA-treated rats given DZX15 was not significantly different from control rats given DZX25 (Fig. 2C). Despite similar degrees of hypotension, plasma VP and OT levels were significantly lower in 6OHDA-treated rats vs. control rats given DZX25 (10 and 30 min; Figs. 2, A and B). A linear regression analysis between plasma VP and OT levels vs. MAP in control rats receiving DZX15 or DZX25 revealed a significant correlation at 10 min (Fig. 3) and 30 min (plot not shown). Interestingly, every 6OHDA-treated rat receiving DZX15 fell outside the 95% confidence intervals of this regression line. That is, 6OHDA-treated rats had significantly lower plasma VP and OT levels for a similar drop in MAP compared with control rats. These differences in plasma VP and OT levels between 6OHDA-treated and control rats cannot be explained by differences in Posmol (Table 1).

Mean ± SE of plasma VP (A) and OT (B) levels and MAP (C) of 6OHDA-treated and control rats administered diazoxide (DZX; 15 or 25 mg/kg iv). Administration of DZX significantly increased plasma VP and OT levels and decreased MAP from baseline values in all groups (P < 0.05). In control rats, the larger dose of DZX produced a significantly larger increase in plasma VP and OT levels as well as a greater drop in MAP at every time (P < 0.05). Administration of DZX15 to 6OHDA-treated rats produced a decrease in MAP similar to those levels in control rats receiving DZX25; however, plasma VP and OT levels were significantly less in 6OHDA-treated vs. control rats receiving DZX25. Although plasma VP and OT levels of 6OHDA-treated vs. control rats receiving DZX15 were not different at 10 min and significantly elevated at 30 min, MAP of 6OHDA-treated rats was significantly lower than those of control rats receiving DZX15 at every time. *Significant difference from control rats receiving DZX15 (P < 0.05). †Significant difference between control rats receiving DZX25 and 6OHDA-treated rats (P < 0.05).

Plasma VP (A) and OT (B) levels at 10 min plotted as a function of MAP for individual rats that received an injection of DZX (15 or 25 mg/kg iv). The MAP values were averaged from 5 and 10 min. A linear regression analysis reflected a significant correlation between plasma VP and OT levels and MAP of control rats receiving DZX (r = 0.72 and 0.81, respectively; P < 0.05). However, plasma VP and OT values of 6OHDA-treated rats receiving DZX15 fell outside the 95% confidence intervals (dashed line). The regression line is the solid line. Similar results were obtained for plasma VP and OT values at 30 min (r = 0.66 and 0.72, respectively; plots not shown).

Effect of 6OHDA Treatment on Hyperosmotic-Evoked Increase in Plasma VP and OT Levels

The infusion of HS significantly increased plasma VP and OT levels and Posmol in both control and 6OHDA-treated rats (Fig. 4). In marked contrast to hemorrhage and hypotension, the increase in plasma VP and OT levels during the infusion of HS was not different between control and 6OHDA-treated rats at 60 and 120 min (Figs. 4, A and B). In addition, Posmol was not different between groups at any time (Fig. 4C). The infusion of HS did not alter MAP (Fig. 4D) or HR from baseline values in 6OHDA-treated vs. control rats (baseline HR: 389 ± 7 vs. 406 ± 11 bpm, respectively).

Mean ± SE of plasma VP (A) and OT (B) levels, plasma osmolality (Posmol; C), and MAP (D) of 6OHDA-treated and control rats infused with hypertonic saline (HS) for 120 min. Infusion of HS significantly increased plasma VP and OT levels from baseline values in both groups (P < 0.05). In marked contrast to serial hemorrhage or DZX-evoked hypotension, the increase in plasma VP and OT levels was not different between groups at 60 and 120 min. Posmol did not differ between groups at any time. As previously reported (29, 31), infusion of HS did not alter MAP in control rats, and there were no differences in MAP values at any time between groups (P > 0.3 from overall ANOVA).

Effect of 6OHDA Treatment on DβH Immunoreactivity in the Posterior Pituitary and Hypothalamic Nuclei

Quantification of DβH immunoreactivity in the posterior pituitary, hypothalamic nuclei and forebrain circumventricular organs of 6OHDA and vehicle-treated rats

The posterior pituitary of vehicle-treated rats contained numerous DβH immunoreactive axons and terminals (Fig. 5A). In contrast, only sparse DβH immunoreactivity was present in the posterior pituitaries of 6OHDA-treated rats (Fig. 5B). The anterior pituitary contained few, if any, DβH-positive fibers in either group and therefore was not scored (data not shown). The ability of 6OHDA treatment to reduce DβH immunoreactivity in the posterior pituitary was in stark contrast to the effect of 6OHDA treatment on the hypothalamic paraventricular and supraoptic nuclei (Fig. 5, C–F). As expected (32), the hypothalamic paraventricular and supraoptic nuclei contained a dense level of DβH-immunoreactivity in vehicle-treated rats (Fig. 5, C and E). In the paraventricular nucleus, DβH-positive fibers were present in both parvocellular and magnocellular divisions throughout its rostral-caudal extent. In marked contrast to its effect on the posterior pituitary, 6OHDA did not alter the level of DβH immunoreactivity in either the paraventricular or supraoptic nuclei (Fig. 5, D and F; Table 2).

Dopamine-β-hydroxylase (DβH) immunoreactivity in the posterior pituitary, hypothalamic paraventricular nucleus, and supraoptic nucleus of 6OHDA and vehicle-treated rats. 6OHDA treatment reduced DβH immunoreactivity in the posterior pituitary (A and B). In marked contrast, 6OHDA treatment did not reduce DβH immunoreactivity in any rostral-caudal level of the paraventricular nucleus, including level 2 with a prominent posterior magnocellular cell group (C and D) or the supraoptic nucleus (E and F). 3V, third ventricle; OC, optic chiasm. Scale bars = 100 μm.

The forebrain lamina terminalis of vehicle-treated rats also contained numerous DβH-immunoreactive axons and terminals (Fig. 6). We observed several DβH-positive fibers in the subfornical organ, the dorsal and ventral median preoptic nucleus, and the organum vasculosum of the lamina terminalis. With regard to the latter, numerous axons and terminalis were located in the lateral and dorsal boundaries with only sparse labeling in the central core (Fig. 6G). 6OHDA treatment did not affect the level of DβH immunoreactivity in these structures except for the subfornical organ (Fig. 6; Table 2).

DISCUSSION

Peripheral catecholaminergic inputs to the posterior pituitary may influence VP and OT release from neurohypophyseal nerve terminals (1, 16, 17, 19, 29), but their role in VP and OT secretion during physiological challenges has received little attention. In the present study, destruction of catecholaminergic nerve terminals located outside the blood-brain barrier with 6OHDA significantly attenuated the increase in plasma VP and OT levels stimulated by hemorrhage and hypotension but not hyperosmolality. These findings suggest that peripheral catecholaminergic fibers contribute to VP and OT secretion during conditions associated with sympathoadrenal activation.

Intravenous administration of the neurotoxin 6OHDA destroys sympathetic catecholaminergic fibers (15, 18). Because 6OHDA cannot cross the blood-brain barrier, catecholaminergic neurons and fibers within the central nervous system and inside the blood-brain barrier should remain largely unaffected in adult rats (15, 20). The present findings provide strong support for this notion as 6OHDA treatment did not reduce DβH immunoreactivity in the hypothalamic paraventricular and supraoptic nuclei. Here, the effectiveness of the 6OHDA treatment to destroy peripheral catecholaminergic fibers was assessed by the magnitude of the tyramine-evoked pressor response, and this pressor response was virtually abolished in 6OHDA-treated rats. Because the ability of tyramine to increase arterial blood pressure depends upon the integrity of presynaptic terminals and norepinephrine concentrations (7, 12), a diminished pressor response would reflect a depletion of terminal noradrenergic stores or destruction of sympathetic nerve terminals. Given the mechanism of action of 6OHDA, it seems likely that the 6OHDA treatment regimen used in the present study destroyed the majority of sympathetic catecholaminergic terminals, consistent with previous observations (7, 18).

The posterior pituitary is innervated by sympathetic catecholaminergic fibers that originate from the superior cervical ganglion (3). This input likely has functional significance as electrical stimulation of the superior cervical ganglion increases VP and OT secretion (16, 17). However, some catecholaminergic input to the posterior pituitary may originate from the central nervous system. For example, lesion of the ventral noradrenergic tract has been reported to reduce norepinephrine levels of the neural lobe (2), and Garten et al. (10) reported that a small number of A2 neurons were labeled after injection of a retrograde tracer targeted at the posterior pituitary. On the other hand, several reports suggest that the superior cervical ganglion, not central catecholaminergic neurons/tracts, provides the vast majority of the catecholaminergic innervation of the posterior pituitary (1, 3, 19). In the present study, 6OHDA treatment markedly reduced DβH immunoreactivity in the posterior pituitary, and this could be attributed to the actions of 6OHDA on catecholaminergic fibers of sympathetic or central origin but located outside the blood-brain barrier. Although 6OHDA treatment did not destroy DβH-positive fibers within the hypothalamic paraventricular and supraoptic nuclei, we did observe a decrease in DβH immunoreactivity within the subfornical organ. This effect is likely explained by the lack of a complete blood-brain barrier at this circumventricular organ; however, noradrenergic innervation of the organum vasculosum of the lamina terminalis was unaffected. The origin of noradrenergic input to the subfornical organ arises from the A1 and A2 cell groups (5, 14, 36), but there is currently no available evidence to suggest that noradrenergic inputs to the subfornical organ contribute to VP and OT secretion. Therefore, it seems likely that the effects of 6OHDA treatment on VP and OT release can be largely attributed to the destruction of peripheral catecholaminergic innervation of the posterior pituitary. The destruction of sympathetic fibers likely contribute to this response, but a contribution from the small number of brainstem neurons that project outside the blood-brain barrier cannot be excluded.

Hemorrhage and arterial hypotension stimulate the secretion of VP and OT. With both stimuli, the increase in plasma VP and OT levels was blunted in 6OHDA-treated rats vs. control rats. The attenuation of VP and OT levels still occurred despite a significantly lower MAP of 6OHDA-treated rats in some instances, which would be expected to produce an even greater stimulus for VP and OT secretion. Although ascending noradrenergic inputs facilitate putative VP cell responses and associated VP secretion in response to hemorrhage/hypotension (4, 11, 25, 26), these inputs are largely unaffected by peripheral 6OHDA administration in adult rats (15, 20). Indeed, the present findings provide strong support for this notion as DβH immunoreactivity was not different in the hypothalamic paraventricular and supraoptic nuclei between 6OHDA and vehicle-treated rats. Therefore, it appears that peripheral catecholaminergic inputs to the posterior pituitary contribute to VP and OT secretion during hemorrhage and arterial hypotension.

The inability of intravenous 6OHDA treatment to affect VP and OT secretion during hyperosmolality is in stark contrast to its ability to blunt plasma VP and OT levels to hemorrhage and hypotension. Hemorrhage and hypotension produce robust increases in sympathetic nerve activity (8, 21, 34), whereas hyperosmolality produces substantially weaker and nonuniform changes in sympathetic outflow (35). For example, Weiss et al. (35) reported that systemic hyperosmolality produced by intravenous infusion of hypertonic saline moderately increases lumbar sympathetic nerve activity but decreases or does not change renal and splanchnic sympathetic nerve activity. In light of the present findings, it is interesting to speculate that the ability of 6OHDA treatment to attenuate VP and OT secretion stimulated by hemorrhage and hypotension, but not hyperosmolality, is related to the effect of each stimulus on sympathetic nerve activity. That is, an increase in sympathetic outflow at the level of the posterior pituitary during hemorrhage and hypotension enhances VP and OT secretion. The lack of an effect of 6OHDA treatment on plasma VP and OT levels in hyperosmotic rats may be due to the osmotic stimulus producing minimal changes in the relevant sympathetic nerves. Future experiments that record activity of sympathetic nerves innervating the posterior pituitary under these various physiological conditions are needed to fully explore this possibility.

In summary, the present findings suggest that peripheral catecholaminergic fibers enhance VP and OT secretion under certain physiological conditions, as systemic administration of the neurotoxin 6OHDA blunted VP and OT levels stimulated by hemorrhage and hypotension, but not hyperosmolality. Because the former two stimuli are associated with pronounced increases in sympathetic outflow, this mechanism might permit the enhancement of VP and OT secretion under conditions in which pronounced activation of multiple pressor mechanisms is needed to maintain arterial blood pressure. The present findings also highlight potential concerns for the use of peripheral 6OHDA administration to elucidate the role of different pressor mechanisms in physiological responses. The results of studies using peripheral 6OHDA treatment to distinguish between the contribution of different pressor systems (sympathetic vs. VP) should be interpreted cautiously, as the present findings clearly demonstrate that systemic 6OHDA treatment eliminates peripheral sympathetic fibers but also blunts VP secretion under conditions of sympathoadrenal activation.

GRANTS

This research was supported by National Institutes of Health Grants HL-55687 (to A. F. Sved), HL-073661 (to S. D. Stocker), HL-073693 (to M. E. Wilson), COBRE Grant P20 RR-015592 (to M. E. Wilson), and a Scientist Development Grant from the American Heart Association (S. D. Stocker). C. J. Madden was supported by a Predoctoral Fellowship from the American Heart Association.

Acknowledgments

The authors thank Kimberly Allred for technical assistance and Dr. John Fernstrom for his generous gift of the VP and OT antibodies.

Footnotes

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.